Genetic Modification of Human Hematopoietic Stem Cells with Pseudotyped rAAV

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DESCRIPTION (provided by applicant): The ideal stem cell gene therapy vector remains as yet unidentified. Unlike other viral vectors for gene transfer into hematopoietic stem cells (HSC), rAAV transduces cells in the G0 phase of the cell cycle, including primitive multipotent HSC making rAAV particularly attractive for evaluation for HSC gene transfer. Recently the human marrow was identified as a common site for native AAV, indicating that it serves as a natural target. The use of novel AAV capsids has in some cases been shown to vastly improve gene transfer efficiencies and overcome limitations in onset of expression. We have previously shown that rAAV2 preferentially transduces primitive HSC residing in GO and capable of multilineage and serial engraftment. Here we propose to evaluate rAAV2 genomes pseudotyped in AAV 1,5,7,8&9 capsids for transduction of primitive human HSCs determine if transduction may be further improved. Our preliminary data indicates that certain serotypes efficiently transduce human HSC capable of long-term engraftment and robust expression in vivo at levels higher than that attained with rAAV2, necessitating the evaluation of pseudotyped rAAV for HSC gene transfer. Transductions will be evaluated in vivo with transduced human HSCs transplanted into immune-deficient mice. The specific aims are: 1. Evaluation of transduction of human HSC capable of longterm multi-lineage primary and serial engraftment by pseudotyped rAAV. Serotypes will be compared for expression, primary and serial reconstitution, safety and toxicity. Differences between capsids with identical genomes will be exploited to dissect steps important for HSC transduction. 2. Identification of chromosomal sites of pseudotyped rAAV integration in HSCs, important to assess genotoxic potential. Comparision of junction sites between pseudotypes will provide insight into mechanisms underlying intgeration site selection. 3. Analysis of insulator function of AAV ITRs in transduced human HSC. We will test our recent exciting discovery of insulator-like functions of AAV ITRs in the context of HSCs. When completed this research should not only identify the most robust and safest AAV serotype for stem cell gene transfer but would also elucidate mechanisms underying the differences between the serotypes. Direct evaluation of human HSC in an in vivo setting will should provide preclinical data directly relevant to any therapeutic application. Delineating insulator function of AAV ITRs in HSC would identify a safe, efficient gene transfer vector for human HSC with a greatly reduced risk of insertional oncogenesis. RELEVANCE TO PUBLIC HEALTH: This research explores a novel gene transfer vector system for safe and effective therapies for blood stem cells. The outcome of this work is expected to identify the optimal gene delivery vector for the treatment of a variety of blood diseases including inherited and infectious diseases.